RIF1 regulates early replication timing in murine B cells

The mammalian DNA replication timing (RT) program is crucial for the proper functioning and integrity of the genome. The best-known mechanism for controlling RT is the suppression of late origins of replication in heterochromatin by RIF1. Here, we report that in antigen-activated, hypermutating murine B lymphocytes, RIF1 binds predominantly to early-replicating active chromatin and promotes early replication, but plays a minor role in regulating replication origin activity, gene expression and genome organization in B cells. Furthermore, we find that RIF1 functions in a complementary and non-epistatic manner with minichromosome maintenance (MCM) proteins to establish early RT signatures genome-wide and, specifically, to ensure the early replication of highly transcribed genes. These findings reveal additional layers of regulation within the B cell RT program, driven by the coordinated activity of RIF1 and MCM proteins.

conversions, resulting in RT with less dynamic range (toward flat RT), especially in primary B cells.This is similar to what has been observed in human iPS cells.Does Rif1 binds selectively to early replicating domains in this chromosome region (chr2: 57,676,011-121,972,628)?It is known, in some cancer cells, that Rif1 binds to selective segments of chromosomes and RT of not all the chromosomes are regulated by Rif1.Do all the early replicating domains respond to Rif1 depletion by causing later replication?Or is there any segmental selectivity in RT regulation by Rif1 in activated B cells?I see the transition of the late replicating domains to earlier-replicating in Rif1 -/-cells (Figure 1D).How is the Rif1 binding profile in those segments, where early replication is not affected or where late replication is converted to early by loss of Rif1? 2 In fission yeast Rif1 mutant, E to L transmission is observed at a number of early replicating regions (Hayano et al. 2012;Genes and Dev), and it has been explained that rate-limiting replication factors are utilized at activated late origins, and cannot be used for early origins, thus reducing origin firing at some early firing origins.A possibility remains that, due to activation of late origins which utilizes initiation factors, early origins cannot be fully activated due to the potential low abundance of initiation factors in activated B cells.This possibility needs to be discussed.
3 Authors state as follows (line 206)."Peaks in both cell types were enriched in early-replicating regions (Fig. 3C-D).However, although RADs in MEFs were mostly late-replicating, as described, in B cells, they were largely early-replicating (Fig. 3C-D)."It is not intuitively clear that RADs (RIF1-associated domains) are late replicating, while Rif1 binding peaks are in early-replicating regions in MEFs.How are the peaks are enriched in the early-replicating regions, but Rif1 associates mainly in the late replicating regions?Some more explanation is needed here for lay people to this field to be able to understand the points.
4 I suggest authors conduct several experiments described below to improve the manuscript before it is further considered for publication in NC.
4-1 Mechanistic insight into how Rif1 may directly regulate early firing origins is lacking in this manuscript.It has been well established that Rif1's ability to recruit PP1 plays a major role in its ability to suppress origin firing.Authors need to examine the PP1 biding mutant of Rif1 in B cells to determine whether PP1 recruitment is required for early origin promotion in B cells.Authors also need to examine the phosphorylation states of MCM4 and Treslin, which are known to be enhanced in Rif1-depleted cells.

4-2 Figure 3E
The data in MEF needs to be shown in comparison.H3K9 methyl pattern also needs to be shown.It has been reported that Rif1 binding sites and H3K9me3 do not overlap.
4-3 Authors propose that Rif1 loss leads to reduced licensing, causing less efficient early origin firing.This possibility is testable by Mcm ChIP-seq, along with the data showing the extent of licensing in Mcm6 knockdown cells.This will also test the authors hypothesis that licensing is lost specifically in highly transcribed regions in the absence of Rif1.

4-4 Figure 5
The extent of Mcm6 depletion in both Rif1+ and Rif1-/-cells need to be shown by western analyses.

Minor comments:
It would be helpful for readers if authors clearly state, in each figure, which cells were used for analyses, primary B cells or CH12 cells.

Fig. 1G
Y axis needs to be explained in the legend.

Fig. 3E
The correlation between Pro-seq peaks and RIF1-ChiP-seq peaks appears not to be very strong.What is the extent of overlap between Pro-seq and Rif1-ChIP peaks?
Reviewer #3 (Remarks to the Author): This manuscript describes the effect of Rif1 on DNA replication in murine B lymphocytes, and reports the surprising finding that Rif1 associates most strongly with early-replicating chromosome domains, and that removal of Rif1 delays the replication of these normally early regions.Numerous previous studies have shown that Rif1 generally specifies late replication in both mouse and human cells, at least partly by directing Protein Phosphatase 1 to dephosphorylate the MCM replicative helicase and possibly other targets.In this study, the authors used the Repli-seq method to examine replication timing (RT) in Rif1-/-mouse CH12 cells and in mouse primary B cells.In contrast to previous findings in other cell lines and to the established model of its action, they find that Rif1 is important for specifying the replication time of the early replicating regions.In some other cell lines Rif1 has been implicated by 3C analyses in specifying chromosome architecture, but in mouse B cells any effect on genome architecture is minor.In a particularly interesting and strong experiment, in Fig. 3 the authors use Repli-seq and ChIP-seq to show that in B cells Rif1 is specifically bound to early domains, in complete contrast to the situation in MEFs where Rif1 associates with late domains as previously reported in the literature.This surprising result certainly gives an entirely new view of how Rif1 may affect DNA replication.However, the authors' proposal that Rif1 directly regulates early origins is weaker: SNS-seq is a controversial method and even accepting the data shown in Fig. 4 at face value, only a few of the very strongest origins are affected.Nor is any data presented indicating that Rif1 interacts specifically or directly with origin sites.Still, the interpretation of this SNS-seq experiment does not affect the main conclusions of the paper concerning Rif1 association domains and effects on timing.The authors go on to show that MCM and Rif1 act additively in B cells to affect early RT and early origin activity, with MCM having a noticeably stronger effect.Perhaps surprisingly, even depleting both MCM and Rif1 does not affect chromatin organisation.Finally, the authors make the interesting observation that in Rif1-/or shMcm6 B cells the regions whose replication timing is most compromised are those that show the highest levels of transcription.Based on this last finding the authors provide an interesting Discussion suggesting reasons why Rif1 might have been re-deployed in B cells to have this special effect on replication, and relate their findings to an earlier study where human Rif1 was shown to stabilise Orc1 on chromatin to promote origin licensing.Overall, given the interesting new findings the paper is a good candidate for Nature Communications.The following points should be addressed: Major Points 1.In places the textual explanations are insufficient and/or opaque for the non-expert.In several places more detailed explanation should be provided explaining the aspects on which the reader should focus (e.g., for Fig. 1C, Fig. 2E and Fig. 3E).2. The Title and Abstract should specify that the study examines mouse cells.3.Although it's clear that the major effect of Rif1 KO is on early regions, there is also some advancement on the replication time of late regions, seen for example in Fig. 1B and 1D.This effect, consistent with previous studies of Rif1, is overly downplayed in the main text.4. Saddle plots in Fig. 2F-G seem to over-represent/exaggerate the changes in chromatin contacts that are seen in Fig. 2E.Therefore, Fig. 2F-G could be omitted.5.The co-localisation of Rif1 peak intensity with transcription, chromatin accessibility and the H3K27ac mark is mild/weak in each case in Fig. 3E.This should be mentioned in the text.6. Fig. 4B uses SNS-seq to identify sites of initiation, which is controversial as the technique may assign some sites of re-priming as origins of replication.Given this issue, the mildness of the effect in Fig. 4B, and the fact that Rif1 does not bind origin sites directly but rather early domains, the authors should alter their abstract to remove the words 'regulates early origin firing', and moderate their claims that Rif1 directly controls origin initiation.7. The meaning of the vertical 'lines' observed through the centre of the DHS-seq and H3K36me3 experimental datasets in Fig. 4B is unclear.Could the authors elaborate on the reason for these low signal zones?8.It would be helpful to move Fig.S4G into Fig. 5 to show the density-contour plots of Rif1-/-and Rif1-/-shMcm6 cells together with their effects on RT and chromatin architecture (using Hi-C).9.The scatter plot in Fig. 6A regarding Rif1-/-shMcm6 cells is not accompanied by the same heatmaps as in Fig. 4B for Rif1-/-cells.This data should be included in Fig. 6. 10.Could the authors test whether the positive effect of Rif1 on early origin firing is dependent on PP1?This would further strengthen their model as outlined in the discussion section.
2. An experimental condition label (i.e., shMcm6 vs. shLacZ) on top of each panel would make it clearer what each experiment is showing (e.g., Fig. 1C).3. Fig. S1A and S1C and Fig. S3A-B show the reproducibility of each Repli-seq, SNS-seq or ChIP-seq experiment, but are not particularly informative for the general reader and could be less prominent.4. The authors could associate an 'Early' or 'Late' label with the positive or negative values in each figure where RT is examined (e.g., Fig. 1B).At least for this Reviewer, the graphs could be read more intuitively with the x-axis reversed so 'Early' is on the left and 'Late' on the right.5.The blue and red colours used in Fig. 1F and 1G are confusing as they don't mean the same thing (i.e., one denotes RT and the other denotes an experimental condition e.g., shMcm6).6.In Fig. 2C, given the fairly minor changes in RT that occur in Rif1-/-conditions (i.e.Early regions somewhat delayed, but still Early) we wouldn't expect much of a change in PC1 values.So, the conclusion drawn is perhaps not as surprising as the authors suggest and is somewhat overstated.7. The lane order between Fig. 3C and Fig. 3D is changed, which is confusing when trying to compare RT and Rif1 localisation in the primary B and MEF cell lines.8.The inclusion of the word 'decreasing' in the y-axis label of Fig. 3E is confusing.The authors should omit this word but keep the grey bar indicating peak intensity.9.A couple of figures are mislabelled in the main text (e.g., Fig. 2F on page 7 should read Fig. 2G).Fig. 4A is not referred to in the main text.10.The data in Fig. 4B is presented with the 'strongest' origins at the bottom, which is confusing, please invert these pile-up plots.11.The black colour used in Fig. 6D to denote overlaps should be changed to be more translucent as it masks the data underneath it.12. Fig. 7A can be omitted as Fig. 7B makes the point in a clearer and more intuitive manner.13.The sentence in lines 385-388 is confusing and should be rewritten to clarify (do the authors mean to say 'earlier' rather than 'later' in line 388?)

Response to reviewers
We would like to thank the reviewers for their time, appreciation of our work and constructive comments on our manuscript.We are happy that all reviewers appreciated our new findings on the role of RIF1 in early RT in B cells.We have incorporated many of the suggestions from the reviewers which have considerably strengthened the manuscript.Please find below our point-by-point response to all comments in blue text.All new text in the main manuscript is also in blue text.

Reviewer #1 (Remarks to the Author):
Regulation replication timing is a critical for appropriate cell division and preservation of genomic integrity.The mechanisms that coordination of early versus late origin firing remain poorly understood.
In this manuscript, the authors investigate the function of RIF1 in regulation of replication timing in activate B cells.They find that RIF1 controls early replicating regions and loss of RIF1 disrupts timing of these early replication sites to a greater extent that late replicating regions.This is a new and unexpected function of RIF1, which has been previously demonstrated to suppress late origins of replication in heterochromatin.The manuscript is well-written with well-controlled and complementary experiments.The authors do a very nice job distilling complex experiments and results into understandable and interpretable findings for readers.The results are novel and establish new insights into replication control in B cells as well as provide foundation for cell type-specific mechanisms for replication timing control.
A few minor comments: 1.In Figures 1 through 5, the green (Rif1-/-) and blue (WT) colors are difficult to distinguish in some of the figures, particularly in the UCSC browser tracings.More distinct color options would make it easier for readers to interpret results.
We have darkened the Rif1 track (from lighter to darker green) to make them more discernible from the blue WT and shLacZ tracks.
Thank you, we have done it.We apologize for the lack of clarity.By "density", we meant SNS-seq-derived read density (RPM) of the ISs, not the density of ISs.We have changed the axis label to "ISs (RPM)".The graphic under the figure shows the five classes based on fold-change of IS RPMs.Hence, class 1 ISs were most active in WT cells whereas class 5 were the least active in WT cells.We have changed the X axis to "Fold-change in IS read density (log2 WT/Rif1-/-)".We hope this is clear.We have also made the changes in the figure legends.5-7, MCM6 has the dominant role in regulation of replication timing compared to RIF1.

In Figures
Loss of MCM6 has more significant impact on replication timing in all the assays.Loss of RIF1 in the MCM6-deficient cells does lead to additional changes supporting that the two proteins are complementary and non-epistatic.However, it should be stated/presented in these result sections that MCM6 has the dominant phenotype.The authors do address this in the Discussion but this information should also be included in the presentation and interpretation of the data in the Results.
We have made this conclusion more explicit in the results section.

Reviewer #2 (Remarks to the Author):
This manuscript investigates the role of Rif1 in activated B cells.The results presented are very unexpected, and potentially present a previously unknown function of Rif1.
Rif1 has been known to suppress the firing of replication origins by binding to chromatin in the vicinity of late-firing origins or in the late replicating regions.In this manuscript, authors show that Rif1 binds to early replicating domains and promotes early replication.The data supporting this conclusion appears to be robust and convincing.The data analyses have been conducted in a professional manner.

Major comments:
1 Looking at the data in Figure 1B, it appears that loss of Rif1 induces both E to L and L to E conversions, resulting in RT with less dynamic range (toward flat RT), especially in primary B cells.This is similar to what has been observed in human iPS cells.Does Rif1 binds selectively to early replicating domains in this chromosome region (chr2: 57,676,011-121,972,628)?It is known, in some cancer cells, that Rif1 binds to selective segments of chromosomes and RT of not all the chromosomes are regulated by Rif1.Do all the early replicating domains respond to Rif1 depletion by causing later replication?Or is there any segmental selectivity in RT regulation by Rif1 in activated B cells?I see the transition of the late replicating domains to earlier-replicating in Rif1 -/-cells (Figure 1D).How is the Rif1 binding profile in those segments, where early replication is not affected or where late replication is converted to early by loss of Rif1?
In B cells, RIF1 is present on all chromosomes indicating that there is no differential binding between chromosomes.Please see the new analysis showing this (Fig. S3B-C).
Based on the scatter plots in Fig 1C for CH12 cells, most of the early bins show delayed replication (note the red stripe consisting of early-replicating bins in Fig. 1C).The results are much more striking in primary B cells (scatter plot in Fig. 1E) where all early-replicating bins show lower RT values in Rif1 −/− cells, implying that all early-replicating bins show later replication in the absence of RIF1.In other words, there are almost no bins where early replication is not affected.This is also apparent in Fig. 1I.
For the Rif1-/-primary B cells, there is indeed E to L and L to E, but in the heterozygous condition, the E to L is very dominant (see also Fig. 1D).We infer from this that the E to L changes are the direct effect of decreased RIF1 (an interpretation supported by the RIF1 ChIP-seq results) and the L to E may be due to the indirect effect of having fewer early-firing origins in the full knockout that leads to advanced firing of normally late origins.Accordingly, the size of the E to L shift in Rif1 −/− is stronger than the L to E shift (Fig. 1D and 1G).
Regarding RIF1 binding in late regions, our HMM analysis showed that L bins advanced their RT but did not acquire positive RT values, but some L-like bins gained positive RT values (Fig. 1E).
When we looked at RIF1 binding in these bins, only ~10% of RIF1 sites were found in L-like bins and even fewer in L bins (Fig. 3D).
2 In fission yeast Rif1 mutant, E to L transmission is observed at a number of early replicating regions (Hayano et al. 2012;Genes and Dev), and it has been explained that rate-limiting replication factors are utilized at activated late origins, and cannot be used for early origins, thus reducing origin firing at some early firing origins.A possibility remains that, due to activation of late origins which utilizes initiation factors, early origins cannot be fully activated due to the potential low abundance of initiation factors in activated B cells.This possibility needs to be discussed.
Thank you.This is an important point and we have mentioned it in the revised Discussion (page 15).
3 Authors state as follows (line 206)."Peaks in both cell types were enriched in early-replicating regions (Fig. 3C-D).However, although RADs in MEFs were mostly late-replicating, as described, in B cells, they were largely early-replicating (Fig. 3C-D)."It is not intuitively clear that RADs (RIF1associated domains) are late replicating, while Rif1 binding peaks are in early-replicating regions in MEFs.How are the peaks are enriched in the early-replicating regions, but Rif1 associates mainly in the late replicating regions?Some more explanation is needed here for lay people to this field to be able to understand the points.
We apologize for this lack of clarity, and we have explained it in the revised version (page 8).
Peaks reflect relatively stronger sites of RIF1 occupancy whereas RADs are larger domains where RIF1 is more broadly distributed or where RIF1 peaks are clustered.Both peaks and RADs constitute bona fide regions of RIF1 binding and are not defined based on location or RT, but strictly using different bioinformatics tools for significant enrichment of signal over background.
Thus, although RIF1 is detected mostly as peaks in B cells (16,043 peaks and 289 RADs), in principle, peaks or RADs could be located anywhere in the genome.
4 I suggest authors conduct several experiments described below to improve the manuscript before it is further considered for publication in NC.
4-1 Mechanistic insight into how Rif1 may directly regulate early firing origins is lacking in this manuscript.
It has been well established that Rif1's ability to recruit PP1 plays a major role in its ability to suppress origin firing.Authors need to examine the PP1 biding mutant of Rif1 in B cells to determine whether PP1 recruitment is required for early origin promotion in B cells.Authors also need to examine the phosphorylation states of MCM4 and Treslin, which are known to be enhanced in Rif1-depleted cells.
We fully appreciate that the mechanism of RIF1 function in B cell RT is not addressed in this study.As we have now discussed more clearly in the paper, the RIF1-PP1-mediated dephosphorylation of MCM4 found in other cells, which is an inhibitory function as it suppresses origin firing, cannot explain the RIF1-dependent firing of some early origins in B cells, which is an activating function.It is more likely that the role of RIF1 at early origins, if PP1-dependent at all, is to facilitate the firing or origins, perhaps akin to the PP2A and PP4-mediated dephosphorylation of replication firing factors reported recently (Jenkinson et al. Mol. Cell 2023).However, we are unaware of any positive function of PP1 in origin firing, and therefore, we do not have any candidate protein whose dephosphorylation status we could test in Rif1 −/− B cells.
However, addressing the potential role of RIF1-PP1 interaction in the regulation of early origin firing is technically extremely complex and, even if successful, would take over a year of work.
RIF1 function is dosage-dependent, and therefore we cannot simply transfect a plasmid encoding for Rif1-DPP1 (a RIF mutant deficient in PP1 binding) to rescue the Rif1 −/− B cells.In the paper from our co-author, Sara Buonomo (Gnan et al.Nature Comm.2021), the authors had to: (1) construct ES cells that carried one HA-tagged knock-in allele (Rif1-FH) and one floxed conditional Rif1 allele, and then target a mini-gene in the Rif1-FH allele to generate the DPP1 allele.
Moreover, they showed that Rif1 hemizygotes already harbored some of the knock-out phenotypes.In summary, to build a system so complex when we do not have candidate substrates of PP1 to examine seems to us to go beyond the scope of this work.
Regarding Mcm4 phosphorylation, as found by our co-author, Sara Buonomo, there are no reliable antibodies to detect phospho-Mcm4 in mouse cells.Importantly, however, please note that based on the explanation above, it is unclear how a suppressive mechanism like the dephosphorylation of Mcm4, even if present, would help to explain the positive regulation of origin firing by RIF1 in B cells.Thus, we propose that the mechanism by which RIF1 regulates early RT is unlikely to be linked to Mcm4-dephosphorylation.However, as mentioned above, it could still work via PP1mediated dephosphorylation of other replication proteins.We have mentioned this in the revised Discussion.

4-2 Figure 3E
The data in MEF needs to be shown in comparison.
We have shown this in the revised version (new Fig. 3F).Thank you for pointing it out.
H3K9 methyl pattern also needs to be shown.It has been reported that Rif1 binding sites and H3K9me3 do not overlap.
We have done the comparison with H3K9me3 ChIP-seq as requested (Fig. 3E-F).
4-3 Authors propose that Rif1 loss leads to reduced licensing, causing less efficient early origin firing.

This possibility is testable by Mcm ChIP-seq, along with the data showing the extent of licensing in
Mcm6 knockdown cells.This will also test the authors hypothesis that licensing is lost specifically in highly transcribed regions in the absence of Rif1.This is a good suggestion, but unfortunately, it is technically unfeasible.We have tried multiple times with several antibodies, but Mcm ChIP-seq has never worked for us.We are aware that this is a problem in the field and that many labs, including our co-authors, have struggled with it.We believe that the problem, in large part, may be due to the underlying biology rather than the antibodies.The current understanding in the field is that MCMs are loaded evenly throughout the genome and can subsequently slide along the DNA without ATP.In this case, different cells have different locations of MCMs at any given point of time, which would average out the signal in population-based analyses like ChIP-seq resulting in poor enrichments over background.

4-4 Figure 5
The extent of Mcm6 depletion in both Rif1+ and Rif1-/-cells need to be shown by western analyses.
Please note that this was already shown in Fig. S1B and Fig. S4A.

Minor comments:
It would be helpful for readers if authors clearly state, in each figure, which cells were used for analyses, primary B cells or CH12 cells.
We have now stated this in the figure legends for all figures, Line 181; Fig2.E-F should be Fig2.E-G, Line 183; Fig2.F should be Fig2.F-G, Line 191; Fig2.E-F should be Fig2.E-G, Thank you for pointing it out, we have made the changes.Lower panel is Fig. 1G Y axis needs to be explained in the legend.
We have done so, thank you.Fig. 3E, The correlation between Pro-seq peaks and RIF1-ChiP-seq peaks appears not to be very strong.What is the extent of overlap between Pro-seq and Rif1-ChIP peaks?
To address this point, also raised by another reviewer, we have provided a more detailed analysis of the RIF1 peaks in Fig. 3E-F and Fig. S3E.

Reviewer #3 (Remarks to the Author):
This manuscript describes the effect of Rif1 on DNA replication in murine B lymphocytes, and reports the surprising finding that Rif1 associates most strongly with early-replicating chromosome domains, and that removal of Rif1 delays the replication of these normally early regions.
Numerous previous studies have shown that Rif1 generally specifies late replication in both mouse and human cells, at least partly by directing Protein Phosphatase 1 to dephosphorylate the MCM replicative helicase and possibly other targets.In this study, the authors used the Repli-seq method to examine replication timing (RT) in Rif1-/-mouse CH12 cells and in mouse primary B cells.In contrast to previous findings in other cell lines and to the established model of its action, they find that Rif1 is important for specifying the replication time of the early replicating regions.In some other cell lines Rif1 has been implicated by 3C analyses in specifying chromosome architecture, but in mouse B cells any effect on genome architecture is minor.In a particularly interesting and strong experiment, in Fig. 3 the authors use Repli-seq and ChIP-seq to show that in B cells Rif1 is specifically bound to early domains, in complete contrast to the situation in MEFs where Rif1 associates with late domains as previously reported in the literature.This surprising result certainly gives an entirely new view of how Rif1 may affect DNA replication.
However, the authors' proposal that Rif1 directly regulates early origins is weaker: SNS-seq is a controversial method and even accepting the data shown in Fig. 4 at face value, only a few of the very strongest origins are affected.Nor is any data presented indicating that Rif1 interacts specifically or directly with origin sites.Still, the interpretation of this SNS-seq experiment does not affect the main conclusions of the paper concerning Rif1 association domains and effects on timing.The authors go on to show that MCM and Rif1 act additively in B cells to affect early RT and early origin activity, with MCM having a noticeably stronger effect.Perhaps surprisingly, even depleting both MCM and Rif1 does not affect chromatin organisation.Finally, the authors make the interesting observation that in Rif1-/-or shMcm6 B cells the regions whose replication timing is most compromised are those that show the highest levels of transcription.Based on this last finding the authors provide an interesting Discussion suggesting reasons why Rif1 might have been re-deployed in B cells to have this special effect on replication, and relate their findings to an earlier study where human Rif1 was shown to stabilise Orc1 on chromatin to promote origin licensing.Overall, given the interesting new findings the paper is a good candidate for Nature Communications.The following points should be addressed: We agree that RIF1 has a very weak effect on regulating origin activity.We have modified the text, abstract and discussion accordingly.We also mention in the revised Discussion that SNS-seq does not detect all origins, but probably only the stronger ones, meaning that there remains the possibility that RIF1 may have a broader role in origin activity.
Major Points 1.In places the textual explanations are insufficient and/or opaque for the non-expert.In several places more detailed explanation should be provided explaining the aspects on which the reader should focus (e.g., for Fig. 1C, Fig. 2E and Fig. 3E).
We apologize for this.We have explained these in more detail in the revised version (see blue text in pages 5, 7, 8 and 9).
2. The Title and Abstract should specify that the study examines mouse cells.
We have made changes to the title and abstract to specify the use of mouse cells.
3. Although it's clear that the major effect of Rif1 KO is on early regions, there is also some advancement on the replication time of late regions, seen for example in Fig. 1B and 1D.This effect, consistent with previous studies of Rif1, is overly downplayed in the main text.
We have explained this better in the revised version (pages 5-6), but in essence, we think that this is an indirect effect of the decreased efficiency of early origins in the KO cells.
4. Saddle plots in Fig. 2F-G seem to over-represent/exaggerate the changes in chromatin contacts that are seen in Fig. 2E.Therefore, Fig. 2F-G could be omitted.
We agree with the reviewer but given that such plots are widely used by the genome architecture community to characterize changes in compartmentalization, we anticipate that several readers would expect to see this, and therefore, we have retained it.
5. The co-localisation of Rif1 peak intensity with transcription, chromatin accessibility and the H3K27ac mark is mild/weak in each case in Fig. 3E.This should be mentioned in the text.
We have provided a more detailed analysis of the heatmap in Fig. 3E using k-means clustering, which better addresses the relationship of RIF1 peaks with transcription and chromatin features.6. Fig. 4B uses SNS-seq to identify sites of initiation, which is controversial as the technique may assign some sites of re-priming as origins of replication.Given this issue, the mildness of the effect in Fig. 4B, and the fact that Rif1 does not bind origin sites directly but rather early domains, the authors should alter their abstract to remove the words 'regulates early origin firing', and moderate their claims that Rif1 directly controls origin initiation.
We have amended the abstract as requested and we have made it explicit in the revised text that RIF1 only regulates ~5% of ISs and hence that it is not a major regulator of origin activity.See also see our response in lines 273-276 above.We have done as requested.9.The scatter plot in Fig. 6A regarding Rif1-/-shMcm6 cells is not accompanied by the same heatmaps as in Fig. 4B for Rif1-/-cells.This data should be included in Fig. 6.
We have provided the heatmap as requested (new Fig, 6B).10.Could the authors test whether the positive effect of Rif1 on early origin firing is dependent on PP1?This would further strengthen their model as outlined in the discussion section.
Please see our explanation for reviewer 2, who also raised this point.We explain why testing the requirement of PP1 is technically very challenging and would take well over a year, if successful.
More importantly, even if we were to find a PP1-dependency, we do not know of any candidate RIF1-PP1 target of dephosphorylation in B cells, which is essential to address mechanistic questions.
Thank you for pointing out, we have corrected this.
2. An experimental condition label (i.e., shMcm6 vs. shLacZ) on top of each panel would make it clearer what each experiment is showing (e.g., Fig. 1C).
Please note that in all figures, the axes labels explicitly state the experimental condition.
3. Fig. S1A and S1C and Fig. S3A-B show the reproducibility of each Repli-seq, SNS-seq or ChIPseq experiment, but are not particularly informative for the general reader and could be less prominent.
We agree that they may not be informative to general readers, but we know that there are many who will want to see such analyses to judge the reproducibility for themselves.Indeed, many papers have such analyses.Hence, we have retained it in the supplemental section where its presence is not disruptive to the flow.4. The authors could associate an 'Early' or 'Late' label with the positive or negative values in each figure where RT is examined (e.g., Fig. 1B).At least for this Reviewer, the graphs could be read more intuitively with the x-axis reversed so 'Early' is on the left and 'Late' on the right.
We generated these plots following the convention in the field (for example, all papers by our coauthors and leading RT experts, D. Gilbert and S. Buonomo, use this layout for showing RT).
Hence, we have chosen to stick to this style.
5. The blue and red colours used in Fig. 1F and 1G are confusing as they don't mean the same thing (i.e., one denotes RT and the other denotes an experimental condition e.g., shMcm6).
Thank you for raising this.We have changed the colors in Fig. 1F and 1H.
6.In Fig. 2C, given the fairly minor changes in RT that occur in Rif1-/-conditions (i.e.Early regions somewhat delayed, but still Early) we wouldn't expect much of a change in PC1 values.So, the conclusion drawn is perhaps not as surprising as the authors suggest and is somewhat overstated.
RT and genome organization are uncoupled, as we and others have shown earlier.Also, RIF1 was shown to have more significant effects on genome organization in other cells.Given this background, it was not predictable that loss of RIF1 in B cells would have the minor effects we saw.In other words, it was theoretically possible that RIF1 could have had a more major effect on genome compartmentalization even if the RT effect was small.7. The lane order between Fig. 3C and Fig. 3D is changed, which is confusing when trying to compare RT and Rif1 localisation in the primary B and MEF cell lines.
We have changed the order in Fig. 3C.
8. The inclusion of the word 'decreasing' in the y-axis label of Fig. 3E is confusing.The authors should omit this word but keep the grey bar indicating peak intensity.
We have changed this heatmap with a new analysis of the same data, as mentioned above.9.A couple of figures are mislabelled in the main text (e.g., Fig. 2F on page 7 should read Fig. 2G).Fig. 4A is not referred to in the main text.
We have corrected this, thank you for pointing it out.10.The data in Fig. 4B is presented with the 'strongest' origins at the bottom, which is confusing, please invert these pile-up plots.
We have flipped the heatmaps as requested.
11.The black colour used in Fig. 6D to denote overlaps should be changed to be more translucent as it masks the data underneath it.
As the reviewer correctly points out, the black proportion of the figure represents the overlapping part of the signal of both tracks whereas the remaining color part shows the excess of one or the other analyzed condition.Therefore, there is no underlying masked data within the black color section (it simply represents the shared amount of the compared tracks).
12. Fig. 7A can be omitted as Fig. 7B makes the point in a clearer and more intuitive manner.
We understand the point, but we feel that Fig. 7A is very important to show how RT of the bins within a given class are affected and to what degree, because all bins are shown separately and the color change reflects the changes across the bins.This is not possible in Fig. 7B where all bins are clubbed together.We do agree with the reviewer that Fig. 7A comes across as the more intuitive analysis, but we feel that both representations are necessary for a comprehensive analysis.
13.The sentence in lines 385-388 is confusing and should be rewritten to clarify (do the authors mean to say 'earlier' rather than 'later' in line 388?) Yes, indeed, we meant to say "earlier".Thank you for noticing it.
The authors have responded to the points raised and certainly adjusted the text and conclusions to take account of the major criticism that the original manuscript text was misleading given the mildness of any direct effect on early origins.
The additional k-means analysis of clustered data in Figures 3E-F is helpful, but in the case of the MEF data shown it is rather confusing whether the RIF1 peaks shown are within/correspond to the (generally late-replicating) RADs present in MEFS, particularly in light of the sentence "In agreement with the fact with most 254 RIF1 peaks in MEFs are in 255 early-replicating regions (Fig. 3C-D)…".So does this plot show analysis specifically of peaks that are not associated with the major RIF1 binding zones in MEFS?
It was disappointing that they dismissed many of the other suggestions that were aimed at clarifying the manuscript to make it more easily understood for the non-expert.
this plot show analysis specifically of peaks that are not associated with the major RIF1 binding zones in MEFS?
It was disappointing that they dismissed many of the other suggestions that were aimed at clarifying the manuscript to make it more easily understood for the non-expert.
We apologize for the confusion, and we have clarified this in the revised manuscript.For the analysis of peaks in Fig. 3C-F, we only define peaks as those that fall outside of RADs.If a peak was called inside a RAD, it is considered as part of that RAD.In other words, peaks and RADs are distinct, non-overlapping regions in our analyses.We have added this explanation to the revised manuscript.In MEFs, the peaks are mostly early-replicating and RADs are mostly late-replicating.
Importantly, both peaks and RADs are bona fide RIF1 binding regions, but their distribution in the genome is different between MEFs and B cells.
Regarding other suggestions, we believed we had addressed all the points made by the reviewer.
For example, we had provided more details for Fig. 1C, Fig. 2E and Fig. 3E, as explicitly mentioned by the reviewer (please see major point 1 from the reviewer in the first review).We went back and checked all our responses, but we did not find anything that was not responded to.
The reviewer had asked us to remove some analyses, such as saddle plots and correlation matrices, but we had also explained why these analyses were retained, namely, because they are widely used and would be expected by many readers.We hope the reviewer understands these points.

REVIEWERS' COMMENTS
Reviewer #2 (Remarks to the Author): Authors responsded to my comments by conducting additional experiments.In Figure S4, authors showed significant decrease in MCM5 enrichment at all the five downregulated ISs, but not at the unchanged ISs.However, this has not been discussed much in terms of effect of Rif1 on early RT.
Although only ~5% of ISs showed >1.5-fold change in read density upon loss of RIF, does this result suggest that Rif1 does affect licensing in activated B cells?And could that be a part of the reason for shift to later replication of early RT in Rif1 KO cells?The culmitative effects of Rif1KO on licensing over the entire genome, whilst the effect at each origin may be small, could affect early replication, since reduced (or delayed) licensing in G1 phase could lead to late firing.I wish authors conducted MCM5 ChIP-seq and examine the correlation between MCM5 binding and Rif1 binding (as well as RT), which would provide more information on potential effect of Rif1 on licensing efficiency and its relation to RT change.(I understand from the letter that the current MCM5 antibody does not work for ChIP seq.)At least authors could discuss the above possibility in the text.
Figure 1B, 2C, 5D, 6D sMcm6 -> shMcm6 3. Line 299 on page 9, text should reference Fig. 4A (rather than Fig. 4B) Thank you, we have changed it.4. Line 242-246, text states "The results showed that in WT cells, class 1 ISs were the most active (Fig. 4C)…whereas class 5 ISs were least active….(Fig.4C-D)."It's not evident that Fig. 4C provides results on which ISs are most versus least active.The graphic under Fig. 4C-D delineates activity of ISs in WT versus Rif1-/-but the data in Fig 4C shows density of ISs in each class, which is not indicative of activity.Can the authors clarify this in the text?

Fig. 5C (
Fig.5C (lower panel) It appears that Rif1-/-advances RT of mid-origins, consistent with other cell types.Authors need to discuss this.

7.
The meaning of the vertical 'lines' observed through the centre of the DHS-seq and H3K36me3 experimental datasets in Fig.4Bis unclear.Could the authors elaborate on the reason for these low signal zones?These low-signal regions are most likely the nucleosome-free regions where the ISs are most abundant.This agrees with previous reports showing that ISs occur in the space between two nucleosomes, which is also where OCCM complexes would reside.8.It would be helpful to move Fig.S4Ginto Fig.5to show the density-contour plots of Rif1-/-and Rif1-/-shMcm6 cells together with their effects on RT and chromatin architecture (using Hi-C).

Figure
Figure 5D legend A magnified view of the boxed region in C. -> There is no box in Figure 5C.